SUMMARY Glycosylation, which fine-tunes the function of proteins, is the most abundant and diverse posttranslational modification. Despite well documented connections to major diseases such as osteoporosis, dyslipidemia, heart disease, chronic obstructive lung disease, cancer, and viral outbreaks, the enzymes that mediate mucin- type O-glycosylation in the Golgi apparatus have yet to be discovered as druggable targets. The initiating enzymes, a family of 20 ppGalNAc-transferase isozymes (herein termed GalNAc-Ts or T1-T20), determine which substrates are modified and at which sites. Significant questions remain regarding their specificity, regulation, targets and functions and the lack of an in situ activity assay and a pharmacological approach have been critical limitations. To address these shortcomings we are developing a panel of isozyme-specific, cell- based fluorescent sensors for GalNAc-transferase activity. They are being used in high-throughput screening to identify isoform-specific, small molecule modulators of ppGalNAc-T mediated O-glycosylation. We will carryout simultaneous screening of compounds with these sensors, which will greatly minimize off-target effects allowing identification of candidates that directly target the enzymes. Preliminary work has resulted in several isozyme-selective biosensors and a few promising drug-like candidates including a remarkably selective inhibitor of T3 that works in both cells and mice with no apparent toxicity. Hits from the screening will be validated using a battery of assays starting with a multi-well format glycosylation assay using purified enzyme preparations that identifies direct-effect compounds. Beyond the scope of this proposal, we plan structural characterization and optimization of any lead compounds as well as initial tests of therapeutic value. Successful identification of isoform-selective modulators promises to be transformative to this area of glycobiology research, and potentially, the clinic.